Saethre-Chotzen syndrome (SCS) is a commonly inherited disorder predisposing humans to premature fusion of the skull calvarial bones (craniosynostosis) and a variety of limb defects. Greater than 45 percent of SCS patients have loss-of-function mutations in one copy of the Twist gene, making Twist haploinsufficiency a causative factor for SCS. Mice also show haploinsufficiency for Twist, as Twist heterozygotes exhibit craniofacial defects and toe duplications. Twist is a well-conserved basic helix-loop-helix (bHLH) protein essential for embryonic development in Drosophila and mouse. Its loss-of-function phenotypes in the limbs suggest that Twist normally has important patterning functions during limb development, interestingly, published and preliminary data show that overexpression of another bHLH protein essential for limb development, HAND2, causes phenotypes resembling those seen in SCS patients and in mice haploinsufficient for Twist. Because HAND2 gain-of-function phenocopies Twist loss-of-function, we hypothesize that HAND2 and Twist interact antagonistically during limb development. We will test for a genetic interaction between the two genes using simultaneous loss-of-function and simultaneous gain-of-function assays in both mouse and chick. In addition, we will use biochemical assays to determine the molecular mechanism behind the interaction between HAND2 and Twist.